Decanoic acid reverse micelle-based coacervates for the microextraction of bisphenol A from canned vegetables and fruits

Decanoic acid reverse micelle-based coacervates were proposed for the extraction of bisphenol A (BPA) from canned vegetables and fruits prior to its determination by liquid chromatography and fluorescence detection at lambda(exc) = 276 nm and lambda(em) = 306 nm. The procedure involved the extraction of minute quantities (300-700 mg) of homogenized food sample with an aqueous solution containing 10% of THF and 0.5% of decanoic acid, conditions under which the coacervate (around 340 microL) formed in situ and instantaneously. The overall sample treatment, which included extraction and centrifugation, took about 25-30 min, and several samples could be simultaneously treated using conventional lab equipment. No clean-up or solvent evaporation were required. Extraction efficiencies mainly depended on the decanoic acid and THF concentration in the aqueous solution and were not affected by the pH or the temperature in the ranges studied (1-4 and 20-60 degrees C, respectively). Recoveries in samples ranged between about 81 and 96%. The precision of the method, expressed as relative standard deviation, was about 3% and the quantitation limit was around 9 ng g(-1), which was far below the current specific migration limit (SML) set for BPA by the EU Commission (600 ng g(-1)). The method was successfully applied to the determination of BPA in the solid content of canned fruit salad, peaches in syrup, mango slices, red peppers, sweetcorn, green beans and peas. BPA was present at concentrations in the range from 7.8 to 24.4 ng g(-1) in canned fruits and from 55 to 103 ng g(-1) in canned vegetables.     

Coacervative extraction of Ochratoxin A in wines prior to liquid chromatography/fluorescence determination

Coacervates made up of reverse micelles of decanoic acid were assessed as a new strategy for the simplification of wine sample treatment in the determination of Ochratoxin A (OTA). Simultaneous extraction/concentration of this contaminant was based on both hydrophobic and hydrogen bond OTA:coacervate interactions. Parameters affecting extraction efficiency and concentration factors were studied. Concentrations of decanoic acid and tetrahydrofuran (THF) were the most influential parameters, being 0.5% of acid and 5% of THF the selected ones. The procedure was very robust, so that the extractions were not influenced by the pH and the nature or concentration of matrix components. OTA recoveries from different types of wines (white, rosé and red) ranged between 85 and 100% and the actual concentration factors varied from 105 to 125 for sample volumes of 15 mL. The detection limits for OTA, after liquid chromatography/fluorimetry (LC/FL) analysis of the coacervate (20 microL), were 4.5 ng L(-1) in white and rosé wines and 15 ng L(-1) in red wines, values which were far below the threshold limit established for OTA by EU directives (2.0 microg L(-1)). No clean-up of the extracts was required for any of the samples analysed. The overall sample treatment took about 15-20 min and several samples could be simultaneously treated using conventional lab equipment. The precision of the method, expressed as relative standard deviation, was about 5%. The approach developed was successfully applied to the determination of OTA in different wine samples from the South of Spain. The concentrations found ranged between 0.015 and 0.091 microg L(-1).     

The Occurrence and Contamination Level of Ochratoxin A in Plant and Animal-Derived Food Commodities

Ochratoxin A (OTA) is a highly toxic mycotoxin and poses great threat to human health. Due to its serious toxicity and widespread contamination, great efforts have been made to evaluate its human exposure. This review focuses on the OTA occurrence and contamination level in nine plant and animal derived food commodities: cereal, wine, coffee, beer, cocoa, dried fruit, spice, meat, and milk. The occurrence and contamination level varied greatly in food commodities and were affected by many factors, including spices, geography, climate, and storage conditions. Therefore, risk monitoring must be routinely implemented to ensure minimal OTA intake and food safety.     

Enzyme-linked immunosorbent assay and colloidal gold immunoassay for ochratoxin A: investigation of analytical conditions and sample matrix on assay performance

A polyclonal antibody against ochratoxin A (OTA) was produced from rabbits immunized with the OTA–BSA conjugate. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a membrane-base colloidal gold immunoassay in flow-through format were developed for the rapid detection of OTA in various food matrices. In the cdELISA, the concentration causing 50% inhibition was 0.07 ng mL⁻¹, and the effects of different chemical conditions (ionic strength, pH value, and organic solvent) were studied. The sensitivity of the assay was higher than those previously reported. A simple, rapid, and efficient extraction method was developed and 74–110% recoveries of spiked samples were obtained. Fifty percent methanol extracts of some food samples such as barley, wheat, oat, corn, rice, and raisins could be analyzed directly by immunoassay after dilution in PBS; grape juice and beer samples could be analyzed directly after dilution with PBS; for coffee samples, a more complex method was used to remove the matrix effect effectively. Membrane-based colloidal gold immunoassays had a visual detection limit of 1.0 ng mL⁻¹ for OTA with a detection time of less than 10 min. For the validation of the cdELISA and membrane-based colloidal gold immunoassay, samples were analyzed by high-performance liquid chromatography. The correlation between data obtained using the microwell assay and HPLC was good (R ² = 0.984). The developed immunoassay methods are suitable for the rapid quantitative or qualitative determination of OTA in food samples.     

Ochratoxin A and 2′R-Ochratoxin A in Selected Foodstuffs and Dietary Risk Assessment

The aim of this study was to estimate the contamination of grain coffee, roasted coffee, instant coffee, and cocoa purchased in local markets with ochratoxin A (OTA) and its isomerization product 2′R-ochratoxin A (2′R-OTA), and to assess risk of dietary exposure to the mycotoxins. OTA and 2′R-OTA content was determined using the HPLC chromatography with immunoaffinity columns dedicated to OTA. OTA levels found in all the tested samples were below the maximum limits specified in the European Commission Regulation EC 1881/2006. Average OTA concentrations calculated for positive samples of grain coffee/roasted coffee/instant coffee/cocoa were 0.94/0.79/3.00/0.95 µg/kg, with the concentration ranges: 0.57–1.97/0.44–2.29/0.40–5.15/0.48–1.97 µg/kg, respectively. Average 2′R-OTA concentrations calculated for positive samples of roasted coffee/instant coffee were 0.90/1.48 µg/kg, with concentration ranges: 0.40–1.26/1.00–2.12 µg/kg, respectively. In turn, diastereomer was not found in any of the tested cocoa samples. Daily intake of both mycotoxins with coffee/cocoa would be below the TDI value even if the consumed coffee/cocoa were contaminated with OTA/2′R-OTA at the highest levels found in this study. Up to now only a few papers on both OTA and 2′R-OTA in roasted food products are available in the literature, and this is the first study in Poland.     

A flow-injection system with amperometric detection for selective determination of antioxidants in foodstuffs and drinks

Amperometric detection in flow-injection systems intended for total determination of antioxidants (flavonoids, oxyaromatic acids, etc.) in various foodstuffs (berries, fruit, vegetables) and drinks (wine, tea, coffee, cognac, beer, etc.) was described and discussed. Advantages of this method were demonstrated. These measurements can underlie building of a bank of data on the content of antioxidants in foodstuffs and drinks.     

Supramolecular solvent-based microextraction of ochratoxin A in raw wheat prior to liquid chromatography-fluorescence determination

A supramolecular solvent made up of reverse micelles of decanoic acid, dispersed in a continuous phase of THF:water, was proposed for the simple, fast and efficient microextraction of OTA in wheat prior to liquid chromatography-fluorescence determination. The method involved the stirring of 300 mg-wheat subsamples (particle size 50 μm) and 350 μL of supramolecular solvent for 15 min, subsequent centrifugation for 15 min and the direct quantitation of OTA in the extract, previous 5.7-fold dilution with ethanol/water/acetic acid (49.5/49.5/1), against solvent-based calibration curves. No clean-up of the extracts or solvent evaporation was needed. Interactions between the supramolecular solvent and major matrix components in the wheat (i.e. carbohydrates, lipids and proteins) were investigated. The reverse micelles in the extractant induced gluten flocculation but only in the coacervation region of lower analytical interest (i.e. at percentages of THF above 11%). The quantitation of OTA was interference-free. Representativity of the 300 mg-wheat subsamples was proved by analysing a reference material. OTA recoveries in wheat ranged between 84% and 95% and the precision of the method, expressed as relative standard deviation, was 2%. The quantitation limit of the method was 1.5 μg kg⁻¹ and was below the threshold limit established for OTA in raw cereals by EU directives (5.0 μg kg⁻¹). The method developed was validated by using a certified reference material and it was successfully applied to the determination of OTA in different wheat varieties from crops harvested in the South of Spain. OTA was not detected in any of the analysed samples. This method allows quick and simple microextraction of OTA with minimal solvent consumption, while delivering accurate and precise data.     

A survey of ochratoxin A and aflatoxins in domestic and imported beers in Japan by immunoaffinity and liquid chromatography.

Analyses of ochratoxin A (OTA) and aflatoxins (AFs) in 94 imported beer samples from 31 producing countries and in 22 Japanese beer samples were performed by immunoaffinity column and reversed-phase liquid chromatography (LC) with fluorescence detection. Recoveries of OTA from beer samples spiked at 25 and 250 pg/mL were 86.1 and 88.2%, respectively. Recoveries of AFs were 98.4 and 98.9%, 95.4 and 95.5%, 101.2 and 97.8%, and 98.9 and 96.0%, respectively, from beer samples spiked at 4.1 and 41 pg AF B1, 4.45 and 44.5 pg AF B2, 4.7 and 47 pg AF G1, and 4.65 and 46.5 pg AF G2/mL. Detection limits were 1.0 pg/mL for OTA, 0.5 pg/mL for AFs B1 and B2, and 1.0 pg/mL for AFs G1 and G2. OTA was detected in 86 (91.5%) of 94 imported beer samples at a mean level of 10.1 pg/mL and in 21 (95.5%) of 22 Japanese beer samples at a mean level of 12.5 pg/mL. AF B1 was detected in 11 of 94 imported beer samples at a level of 0.5-83.1 pg/mL and in 2 of 22 Japanese beer samples at 0.5 and 0.8 pg/mL. Except for one beer sample from Peru, the samples contaminated with AFs were also contaminated with OTA. Although OTA was detected in most samples from various countries, AFs were detected in the beer samples from only a limited number of countries where AF contamination might be expected to occur because of their warm climate.     

Certification of reference materials for ochratoxin A analysis in coffee and wine

Mycotoxins are important non-anthropogenic food and feed contaminants, which can be present on almost every agricultural commodity. Effective consumer protection therefore essentially depends on food surveillance by reliable quantitative analysis enabled by appropriate quality control. Certified (matrix) reference materials (CRMs) are versatile tools to support quality assurance. However, in the case of ochratoxin A (OTA), a hepato- and nephrotoxic mycotoxin, which is regulated in various foods, there is a lack of suitable CRMs. This lack has now been overcome by the development of two European Reference Materials (ERM^?) for the determination of OTA in roasted coffee (ERM^?-BD475) and red wine (ERM^?-BD476). This article discusses the material preparation process as well as the results of homogeneity and stability testing. Furthermore, the results of the in-house certification studies carried out at BAM Federal Institute for Materials Research and Testing are presented and discussed. Interlaboratory comparison studies involving selected expert laboratories with documented expertise in the field of mycotoxin analysis were conducted to confirm the certified values determined by BAM. The certified ochratoxin A values and their corresponding expanded uncertainties (k?=?2) were assigned in full compliance with the requirements of ISO Guide 35 and are as follows: (6.0?±?0.6)???g?kg^?1 for roasted coffee, ERM^?-BD475, and (0.52?±?0.11)???g?L^?1 for red wine, ERM^?-BD476.     

Molecularly imprinted polymer‐based solid phase clean‐up for analysis of ochratoxin A in beer,red wine,and grape juice

A simple, reliable, and low‐cost method based on molecularly imprinted polymer as a selective sorbent of SPE was proposed for the determination of ochratoxin A (OTA) in beer, red wine, and grape juice by HPLC coupled with fluorescence detection (HPLC‐FLD). Samples were diluted with water and cleaned up with an AFFINIMIP® SPE OTA column. After washing and eluting, the analyte was analyzed by HPLC‐FLD. Under the optimized conditions, LOD and LOQ for OTA were 0.025 and 0.08 ng/mL, respectively. The recoveries of OTA from beer, red wine, and grape spiked at 0.1, 2, and 5 ng/mL ranged from 91.6 to 101.7%. Furthermore, after a simple regenerated procedure, the molecularly imprinted polymer based SPE column could be reused at least 14 times to achieve more than 80% recoveries of OTA in real samples. The developed method was applied to the detection of 30 beer, red wine, and grape juice samples and only four samples were contaminated by OTA with levels below the legal limits.     

Synthesis of the Stationary Phase IS-Anionic (Internal Surface-Anionic) for Extraction of Ochratoxin A and B from Samples of Beers

A new IS-anionic stationary phase was synthesized to make on-line extraction of the ochratoxin A and B from samples of beer for HPLC. The propyltriethylammonium chloride stationary phase was characterized affecting it's elementary determination and RI specter, respectively. Evaluation of the IS-anionic column for the extraction and quantification of OTA and OTB in beer has shown that the column is suitable for efficient extraction (recovery >76.5%) and precise analysis. The detection limits for OTA and OTB were 0.03 and 0.07 mugL(-1), respectively. The range of detector linearity was 0.03 at 20 mugL(-1).     

Novel gel-based rapid test for non-instrumental detection of ochratoxin A in beer

A rapid easy-to-use immunoassay was optimised for the non-instrumental detection of ochratoxin A (OTA) in beer. The analytical method involves preconcentration on the immunoaffinity layer inside a column followed by direct competitive ELISA detection in the same layer. The visual cut-off value, i.e. the lowest OTA concentration resulting in no colour development, was 0.2 μg L^-1. Assay validation was performed using samples spiked with OTA. Thirty-seven naturally contaminated samples were screened with the gel-based method developed and no false-negative results were obtained. The method described offers a simple, rapid and cost-effective screening tool, thus contributing to better health protection of consumers. Figure Gel-based immunoassay of spiked beer samples.     

Effect of β-cyclodextrin on spectroscopic properties of ochratoxin A in aqueous solution

Ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by several fungal species, mainly Aspergillus ochraceus, A. carbonarius and Penicillium verrucosum. It contaminates many foodstuffs, particularly cereals and their derivatives, coffee, beer, wine and cocoa, and represents a serious health threat both to humans and animals. Spectroscopic properties of OTA solutions depend on the pH, solvent polarity and can be influenced by the presence of cyclodextrins (CDs). In this work, the effect of β-CD on spectroscopic properties of OTA in aqueous solutions has been investigated by means of absorption and steady-state fluorescence at different pHs (range 3.5–9.5). Binding constants of OTA/β-CD inclusion complexes have been determined by applying modified Benesi-Hildebrand equation. A 1:1 stoichiometry of OTA/β-CD complexes has been observed at all tested pHs.     

Highly Sensitive Impedimetric Biosensor Based on Thermolysin Immobilized on a GCE Modified with AuNP-decorated Graphene for the Detection of Ochratoxin A

The fabrication of a thermolysin-based biosensor capable of detecting ochratoxin A (OTA) from food samples is described. The electrochemical deposition of calcium cross-linked cellulose film (CCLC) and gold nanoparticles (AuNPs) on graphene (GR) for modification of a glassy carbon electrode (GCE) is the first step. Then the thermolysin (TLN) enzyme in a polyvinyl alcohol (PVA)/polyethylenimine (PEI) matrix is immobilized. The impedimetric biosensor response is linear from 0.2 nM to 100 nM with a detection limit of 0.2 nM. The obtained stable and reproducible biosensor is then applied for the detection of OTA in spiked extracts from coffee beans.     

Direct detection of OTA by impedimetric aptasensor based on modified polypyrrole-dendrimers

Ochratoxin A (OTA) is a carcinogenic mycotoxin that contaminates food such as cereals, wine and beer; therefore it represents a risk for human health. Consequently, the allowed concentration of OTA in food is regulated by governmental organizations and its detection is of major agronomical interest. In the current study we report the development of an electrochemical aptasensor able to directly detect trace OTA without any amplification procedure. This aptasensor was constructed by coating the surface of a gold electrode with a film layer of modified polypyrrole (PPy), which was thereafter covalently bound to polyamidoamine dendrimers of the fourth generation (PAMAM G4). Finally, DNA aptamers that specifically binds OTA were covalently bound to the PAMAM G4 providing the aptasensor, which was characterized by using both Atomic Force Microscopy (AFM) and Surface Plasmon Resonance (SPR) techniques. The study of OTA detection by the constructed electrochemical aptasensor was performed using Electrochemical Impedance Spectroscopy (EIS) and revealed that the presence of OTA led to the modification of the electrical properties of the PPy layer. These modifications could be assigned to conformational changes in the folding of the aptamers upon specific binding of OTA. The aptasensor had a dynamic range of up to 5 μg L⁻¹ of OTA and a detection limit of 2 ng L⁻¹ of OTA, which is below the OTA concentration allowed in food by the European regulations. The efficient detection of OTA by this electrochemical aptasensor provides an unforeseen platform that could be used for the detection of various small molecules through specific aptamer association.     

Electrophoretic methods in the analysis of beverages

A review of the applications of electrophoresis to the determination of various compounds in beverage samples, namely beer, hard drinks, juice, milk, soft drinks, tea and wine, is presented.     

Restricted access supramolecular solvents for sample treatment in enzyme-linked immuno-sorbent assay of mycotoxins in food

A restricted access supramolecular solvent (SUPRAS-RAM) made up of tetradecanoic acid reverse micelles is proposed as a wide-scope and low-cost strategy for the treatment of agrifood samples prior to enzyme-linked immunosorbent assays (ELISA). The approach was assessed for the determination of ochratoxin A (OTA) in wines and spices and aflatoxin B1 (AFB1) in cereals, two ubiquitous mycotoxins that were selected as representative contaminants for this study. The samples were selected to cover a variety of matrices in terms diverse composition and high complexity. Macromolecules such as proteins and carbohydrates were not-co-extracted due to the restricted access properties of the SUPRAS that are provided by chemical and physical mechanisms. In this sense, analyte extraction and clean-up were carried out in a single step. Parameters determining the extraction efficiency were studied and optimized. Certified reference materials were used for method validation. Recoveries of OTA ranged between 83% and 96% in wines (with relative standard deviation, RSD, of about 10%) and between 81% and 93% in spices (RSD 7%). Recoveries for AFB1 in wheat ranged from 75% to 85% (RSD 8%). The detection limits were all below the maximum levels established for OTA and for AFB1 by EU directives. This method offers a green and low-cost alternative to the organic solvent-based extraction and/or immunoaffinity columns-based cleanup of complex samples prior to ELISA.     

Electrochemical Aptasensor for the Determination of Ochratoxin A at the Au Electrode Modified with Ag Nanoparticles Decorated with Macrocyclic Ligand

An electrochemical aptasensor for ochratoxin A (OTA) detection has been developed on the base of a gold electrode covered with electropolymerized neutral red and silver nanoparticles obtained by chemical reduction with macrocyclic ligands bearing catechol fragments. Thiolated aptamers against OTA were covalently attached to silver nanoparticles via Ag? S bonding. The interaction with OTA induced the conformational switch of the aptamer, which caused increase of the charge transfer resistance measured by EIS in the presence of ferricyanide ions. The LOD achieved (0.05 nM) was comparable to other electrochemical aptasensors employing sophisticated assembling technique and enzyme amplification of the signal. The aptasensor was validated in spiked beer samples. The recovery of the OTA determination was found to be 66.3±14.1 % for light beer and 64.3±1.8 % for dark beer.     

The comparison of two clean-up procedures, multifunctional column and immunoaffinity column, for HPLC determination of ochratoxin A in cereals, raisins and green coffee beans

To evaluate a clean-up method of detecting ochratoxin A (OTA) by HPLC, the performances of two different clean-up columns, an immunoaffinity column and a multifuntional column were compared in an inter-laboratory study. As samples, un-contaminated wheat, corn grits, green coffee beans and naturally contaminated raisins were used. The recovery test was performed at two different concentrations of OTA (0.5 and 5.0 μg/kg) except for naturally contaminated raisins. Using the immunoaffinity column, the recovery rates, and relative standard deviations for repeatability (R.S.D._r) and reproducibility (R.S.D._R) for wheat, corn grits and green coffee beans ranged 59.0-85.8, 4.2-7.8 and 22.9-29.2%, respectively. For naturally contaminated raisins, recovery, R.S.D._r and R.S.D._R were 84.1, 1.8 and 5.1%, respectively. Using the multifunctional column, the recovery rates, R.S.D._r and R.S.D._R for wheat, corn grits and green coffee beans ranged 80.8-185.0, 0.7-6.9 and 15.2-33.9%, respectively. For naturally contaminated raisins, the recovery, R.S.D._r and R.S.D._R were 128.7, 1.1 and 3.7%, respectively. The results suggest that a multifunctional column could be used to detect OTA in wheat and corn grits at a concentration as low as 0.5 μg/kg; however, it was difficult to detect OTA in green coffee beans and raisins at such a low level. Although an immunoaffinity column could be used for all the test samples in this study from a low level to a high level, the recovery rates were lower than with a multifunctional column.     

Studies on the stability of acrylamide in food during storage

Acrylamide levels in a variety of food samples were analyzed before and after 3 months of storage at 10 degrees-12 degrees C. The analysis was performed by liquid chromatography tandem mass spectrometry (LC/MS/MS) using deuterium-labeled acrylamide as internal standard. Acrylamide was stable in most matrixes (cookies, cornflakes, crispbread, raw sugar, potato crisps, peanuts) over time. However, slight decreases were determined for dietary biscuits (83-89%) and for licorice confection (82%). For coffee and cacao powder, a significant decrease occurred during storage for 3 or 6 months, respectively. Acrylamide concentrations dropped from 305 to 210 microg/kg in coffee and from 265 to 180 microg/kg in cacao powder. On the contrary, acrylamide remained stable in soluble coffee as well as in coffee substitutes. Reactions of acrylamide with SH group-containing substances were assumed as the cause for acrylamide degradation in coffee and cacao. Spiking experiments with acrylamide revealed that acrylamide concentrations remained stable in baby food, cola, and beer; however, recovery levels dropped in milk powder (71%), sulfurized apricot (53%), and cacao powder (17%). These observations suggest that variations in the acrylamide content of food, especially in coffee and cacao, can vary depending on the storage time because special food constituents and/or reaction products can affect the levels.